Bi-media dip plate

ABSTRACT

A BI-MEDIA DIP PLATE FOR USE IN PERFORMING A METHOD OF BACTERIAL QUANTITATION AND DIFFERENTIATION. THE DIP PLATE HAS A RAISED BORDER ON EACH OPPOSED FACE FOR SEPARATING FINITE AREAS OF DEPOSIT FOR CULTURE MEDIA FROM POTENTIAL CONTAMINATNS IN CONTACT WITH THE EDGES OF THE DIP PLATE AND HAS LEGS FOR SPACING THE AREA OF DEPOSIT ABOVE A SUPPORTING SURFACE. THE DIP PLATE IS USED IN COMBINATION WITH A VIAL WHICH MAINTAINS THE DIP PLATE IN A STABLE, ERECT POSITION.

June 18, 1974 D. R. WARREN 3,817,339

BI-MEDIA DIP PLATE Filed March 29, 1971 Mb 200. I0

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F 2 INVENTOR. I

DON R. WARREN United States Patent Office 3,817,839 Patented June 18,1974 3,817,839 BI-MEDIA DIP PLATE Don R. Warren, 715 S. Few St.,Madison, Wis. 53703 Filed Mar. 29, 1971, Ser. No. 128,905 Int. Cl. C12b.l/; C12k N04 US. Cl. 195-140 1 Claim ABSTRACT OF THE DISCLOSUREBACKGROUND OF THE INVENTION (1) Field of the invention This inventionpertains generally to devices for supporting culture media for use inperforming methods of screening for the presence of bacteria, and moreparticularly to a bi-media dip plate for use in performing a method ofbacterial quantitation and differentiation.

(2) Description of the prior art Chemical methods of detectingbacteriuria and the like have been preferred by doctors and hospitalsbecause of the speed with which the test may be completed. However, suchmethods suffer from a high percentage of false-negative results whichseriously impair their usefulness for screening patients.

Other methods such as the pour plate colony count or the use ofcalibrated loops have not been accepted because of the additional timeand labor required by the older methods. Quantitative methods requireprompt shipment of samples to the laboratory in order to obtain anaccurate reading.

A method of diagnosing microorganisms is disclosed in US. Pat. No.3,368,549. A diagnostic swab coated with a culture medium is brought incontact with an area of the anatomy of suspected infection. Theinnoculated swab is transferred to a test tube and incubated in anatmosphere conducive to the growth of the suspected micro-organism. Theculture medium contains inhibitors to prevent the growth of any otherorganisms which might be mistaken for the specific micro-organism beingtested for. a

A similar method has been adopted for use in testing for bacteriuria.One such diagnostic swab is coated with a culture medium such asEosin-Methylene Blue (EMB) agar which inhibits the growth ofcontaminating bacteria. A second swab is coated with a culture mediumsuch as nutrient agar which will support the growth of contaminatingbacteria as well as urinary tract supported organisms.

Both swabs are inoculated with urine and placed in separate vials forincubation. A comparison of the two swabs will indicate whether anygrowths on the EMB agar are a result of the presence of bacteriuria, ormay be due to contaminating bacteria. A comparison of the EMB agar swabto various photographic reference standards indicates the quantitativebacteria growth.

Unfortunately the cost of performing the test and the time required tocomplete it are quite substantial because two separate swabs, twoincubating vials, and two separate inoculations must be performed tocomplete one test.

A slide culture method of bacteriuria has been devised using ordinaryglass microscopic slides. The method is basically the same as the dualswab method except that the two differentiating culture media are placedon either side of a single glass slide. Only one inoculation is requiredto inoculate both media and incubation is carried out in a single vial.Thus there is a saving in time and expense.

Placing both culture media on a single slide has produced many newproblems. Condensation and excess test specimen which collects in thebottom of the incubating vial may reinoculate the culture media toadversely affect the test result. Also condensation collecting at theedges of the glass slide can dissolve the water soluble dyes in the EMBagar. These dyes may then diffuse to contaminate the nutrient agar, orother culture medium, on the opposing face of the slide. This results inno differentiation between the bacteria growth on the test medium andthat on the control medium. A final problem is that it is diflicult tovisually distinguish the bacteria growth on one side of the glass slidefrom the growth on the other side. This of course is because both theglass slide and the culture media are transparent. As a result it isdiflicult to make a quantitative determination of the bacteria growth onthe test medium.

A spacer is often included in the bottom of the incubating vial toprevent reinoculation of the test media. However, the spacer may adhereto the bottom of the glass slide during incubation. Care must beexercised when removing the slide so that the spacer does not flip up asthe slide is retracted to contaminate the media and spoil the test justbefore it is completed.

SUMMARY OF THE INVENTION I have invented an improved bi-media dip platefor use with the slide culture method of bacteria quantitation.

My dip plate is fabricated from plastic and has a raised border on eachface encompassing a finite area thereon for the deposit of a culturemedium. The raised border spaces and isolates the culture media from thejuncture of the edge of the dip plate and the wall of the incubatingvial. Such spacing effectively separates the culture media from contactwith any condensate forming on the abutting wall of the vial. This inturn prevents solubilizing and subsequent contamination of the testmedia by the condensate.

The dip plate also has a pair of legs extending downwardly from thelower edge of the plate. When the inoculated dip plate is placed in theincubating vial the legs space the culture media above any condensationor excess test specimen that may drain into and accumulate at the bottomof the vial. The media are safe from reinoculation and there is nocumbersome or awkward spacer needed.

The dip plate can be transparent or opaque. It can also be manufacturedin any color. This makes it possible to employ numerous combinations ofdifferent culture media, selecting a dip plate in a color appropriate toprovide a proper optical contrast 'between the dip plate and thebacteria growth. Also the problem of confusing the bacteria growth onone side of the dip plate with that on the other side is eliminated byuse of a non-transparent dip plate in almost all cases.

Further objects and advantages will become apparent from the followingdetailed description together with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS- FIG. 1 is a perspective view of a dipplate exemplifying my invention.

FIG. 2 is a side elevation of the dip plate of FIG. 1 with culture mediaapplied thereon.

3 FIG. 3 is a front elevation of the dip plate of FIG. 2 enclosed withina capped cylindrical incubating vial which is shown in section.

DESCRIPTION OF A PREFERRED EMBODIMENT Referring now more particularly tothe drawings my dip plate is shown generally at 10.

The dip plate is an elongated sheet of high pressure molded high impactpolystyrene. It is produced in any color depending on the opticalcontrast required between the culture media and the bacterialcolonization thereon. For urine cultures, for example, a black coloreddip plate 10 is preferred to prevent the reading of colony counts on oneside upon observation from the other side. The colony counts may be readeasily with reflected light. For throat cultures, on the other hand, thedip plate 10 is made of transparent polystyrene because hemolysis of thered blood cells may be observed more easily with transmitted light.

A margin is allowed at the top of the dip plate 10 as a handle 14 forgrasping the plate during inoculation and evaluation. The handle 14 alsoprovides a space on each face of the dip plate 10 for labeling orimprinting selected indicia for promotional or informational purposes.For example one face might be labeled T.'S. Agar to advise the user thatthe culture medium coated thereon is tryptic soy agar, as illustrated inFIG. 1.

Raised borders 11a and 11b on opposite faces of the dip plate 10encompass finite areas 12a and 12b for the deposit of culture medium.The raised borders 11a an 11b have a width of approximately .047 inches.Side borders 11a have a height of .015 inches, whereas end borders 11bhave a slightly lower height of .012 inches to facilitate handling ofthe dip plate during application of the media. [Finite areas 12a and 12bare one inch wide and two inches long.

The function of the raised borders 11a and 11b is to preventcontamination of the culture media on the finite areas 12a and 12b. Theraised borders 11a which extend downwardly along the sides of the dipplate are spaced inwardly a uniform distance along their length from theside edges of the dip plate and thus uniformly space the finite areas12a and 12b away from the inside wall of the incubating vial 20 toseparate the culture media from condensate which may form on the wall.The separation prevents such condensate from dissolving andcontaminating the culture media. The raised side borders 11a shouldprovide a minimum spacing between the finite areas 12a and 12b and theadjacent edges of the dip plate of approximately .075 inches. Inaddition, the raised borders confine the culture media to the finite.areas 12a and 12b during application of the media to the dip plate 10,and during transportation and storage of the coated plate.

A pair of legs 13 extend downwardly from the bottom edge of the dipplate 10. The legs 13 space the dip plate 10 above any contaminants onthe surface supporting the dip plate 10. Typically this surface will -bethe lower end 20a of the cylindrical vial 20 in which the dip plate 10is stored during the incubation period of the bacteria. Excess testspecimen and condensate will drain oif the dip plate and onto thesupporting surface where such materials will not contaminate theinoculated media on the dip plate. The legs 13 should be of sufiicientlength to provide a space of approximately .125 inches between thebottom edge of the dip plate 10 and the principal surface of the viallower end 20a.

It should be noted that incorporation of a spacing means on the dipplate 10 eliminates the need for a separate spacer element in the bottomof the incubating vial 20. Elimination of the spacer reduces the cost ofmanufacture and eliminates any hazard of contamination caused by atipping of the spacer element.

During the incubation period the dip plate 10 is stored in thecylindrical incubating vial 20 shown in section in FIG. 3. The dip plate10 is in close contact with the walls of the incubating vial 20 and itis secured snugly in position by contact with the lid 21 which closesthe open upper end 20b of the vial 20. Together the vial 20 and the lid21 hold the dip plate 10 in a fixed, substantially vertical positionduring incubation. If the dip plate 10 were tipped, movement of liquidfrom one media to the other would be facilitated, with resultingcontamination.

The culture media to be applied to the dip plate 10 will be determinedby the particular bacterial test to be performed. In this respect anytwo culture media which will differentiate between the bacteria beingtested for and bacteria present because of contamination will do. Asmentioned above the dip plate 10 can be manufactured of transparentpolystyrene or of opaque polystyrene in any of a multitude of colors. Byproper choice the best optical contrast between the dip plate 10 and thebacteria growth can be determined. As a result determining thequantitative bacteria colony count on the test medium is greatlyfacilitated.

To provide a bi-media dip plate for urine analysis, for example, it ispreferred to coat the finite area 12a of the dip plate 10 withEosin-Methylene Blue (EMB) agar 15, shown in FIGS. 2 and 3. The dyespresent in the EMB agar inhibit the growth of bacteria other thaninfectioncausing gram negative rods. Bacteria growth on the EMB,therefore, will indicate the presence of the infectious pathogens beingtested for.

The finite area 12b on the other face of the dip plate 10 is coated withTryptic Soy (T.S.) agar 16, shown in FIG. 2. The TJS. agar provides amedium upon which the common contaminants such as diptheroids,staphylococci and streptococci, as well as the infectious pathogens willgrow.

It is preferred that the dip plate 10 be black for use in urineanalysis. The bacteria colonies that grow on the media in urine analysiswill be easily observable by reflected light against the black dip plate10. Also there is no possibility of confusion due to observation ofbacteria colonies on the opposite side of the plate as may occur with atransparent glass slide.

The clip plate 10 may be of transparent manufacture, however, if thetest conditions warrant it. For example a bi-media dip plate for throatculture testing would be prepared with TS. Agar as the control medium onthe finite area 12b and Tryptose blood agar as the test medium on thefinite area 12a. A positive reaction on the Typtose blood agar ishemolysis of the red blood cells which can more easily be observed bytransmitted light. Thus the dip plate 10 should be transparent forthroat culture testing.

In use inoculation and incubation of the culture media will besubstantially similar for most bacterial tests. For example the dipplate 10 discussed above for use in a urine analysis would beinnoculated by dipping it into the urine specimen. When only a smallvolume of samplc is available, the culture media may be inoculated bypouring the specimen directly over the entire surface of each medium.The medium may be looped or streaked with specimen if conditions sorequire.

After inoculation excess sample is drained off and the dip plate 10 isplaced upright in the incubation vial 20. The lid 21 is placed on thevial 20 to secure the dip plate 10 snugly in place. The sample isincubated at least overnight (18 hours) at 37 C. or for 24 hours at roomtemperature.

After incubation the opposing sides of the dip plate 10 are compared totest for contamination. A difference between the bacteria counts on theTS. agar and the EMB agar suggests that the specimen was contaminatedduring the collection process and that a high colony count on the 'EMB,therefore, may not be meaningful. However, abuandant growth on nutrientagar with scant or imperceptible growth on EMB suggests infection withan organism other than gram-negative rods, particularly if the growthseems to be primarily of one colony type. A reliable test is indicatedby substantial uniformity between the colony counts on both sides of thedip plate 10. This indicates that little or no contaminating bacteriaare present.

It should be noted that although the dyes in the EMB agar suppress orinhibit the growth of bacteria that might be mistaken for the presenceof infectious bacteria, the 'EMB supports growth of gram-negativeorganisms. These other organisms however have distinctive features whichthe technician in this field will recognize and easily identify. Theseorganisms therefore need not be discussed here.

Quantitation of the bacteria growth on the EMB in a test determined tobe reliable is performed by comparing the colony density on the EMB withstandardized full scale photographs of serially diluted bacterialsuspensions. These suspensions of standardized counts are confirmed bythe accurate but slower pour plate culture method.

The colony count per millimeter of urine can be quickly calculated fromthe colony count on the slide. For example, if the dimensions of theraised borders 11a and 12a are one inch by two inches, the agar surfacescoated therein upon the finite areas 12a and 12b will each total twosquare inches. Such an area will absorb between .01 and .02 millimetersof urine. The colonization per millimeter of specimen is thereforecalculated to be the colonization per finite area 12a or 12b, times aconstant of 75, which constant is an average between 1 1 m (100) and(50).

Since it is the surface area of the agar that determines specimenretention, it is unnecessary to apply precisely the same quantity ofagar to all dip plates. For example, a normal media thickness of .125inches may be varied by as much as 10% with only an insignificantvariance in surface area. The quantitation method is accurate up toabout 10' colonies per millimeter.

Although the foregoing description of a preferred embodiment isnecessarily of a detailed character in order that the invention may becompletely set forth, it is understood that my invention embraces allsuch modified forms thereof as come within the scope of the followingclaim.

I claim:

1. A bi-media dip plate for use in performing a method of bacterialquantitation and differentiation, said dip plate having a pair ofopposed surfaces and a pair of side edges, a raised border on each saidsurface, each of said raised borders comprising a pair of side bordersand a pair of end borders enclosing a finite area on one of saidsurfaces for the deposit of a culture medium, said side borders beingspaced inwardly from the side edges of said dip plate, said side bordersbeing raised at least .015 inch high and spacing said finite areas atleast .070 inch from the side edges of said dip plate, and said endborders being raised approximately .003 inch less than said sideborders.

References Cited UNITED STATES PATENTS 3,563,859 2/1971 Fink -1393,651,926 3/1972 Elfast 195127 UX ALVIN E. TANENHOLTZ, Primary ExaminerUS. Cl. X.R. 195-139

